Cellular neuroscienceBeta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons
Section snippets
Preparation of hippocampal cultures
Embryonic day (E) 18 rat embryos were used to prepare primary hippocampal cultures as previously described (Goslin and Banker, 1991). Briefly, hippocampi were dissected and freed of meninges. The cells were dissociated by trypsinization followed by trituration with a fire-polished Pasteur pipette. For electron microscopy and subcellular fractionation experiments, hippocampal neurons were plated at high density (500,000 cells/60-mm dish) in MEM with 10% horse serum (MEM10). After 2 h, the medium
Aβ caused accumulation of amphiphysin at the membrane of stimulated cultured hippocampal neurons
We have previously shown that Aβ induced dynamin 1 depletion in cultured hippocampal neurons. An expected consequence of dynamin 1 depletion is the disruption of synaptic vesicle endocytosis during synaptic activity. To obtain evidence of such Aβ potential effects, we used Western blot analysis to detect membranous accumulations of proteins involved in synaptic vesicle endocytosis in control hippocampal neurons and in neurons stimulated in the presence of high potassium stimulation for 10 min.
Discussion
The results presented herein indicated that Aβ impaired synaptic vesicle endocytosis, and hence the normal recycling of these organelles, during sustained synaptic activity in cultured hippocampal neurons. In addition, our data provided further evidence suggesting that these Aβ effects could be mediated, at least in part, by the significant depletion of dynamin 1 induced by this peptide. Together, these findings identify potential mechanisms underlying synaptic dysfunction in early stages of AD.
Acknowledgments
This study was supported by grant NIH/NS39080 to A.F. B.L.K was supported in part by NIA/AG20506 training grant and an American Foundation for Aging Research Fellowship. We thank Rachel Bergstrom and Roxanna Sinjoanu for excellent technical support. We also thank Linda Juarez (RRC EM facility at University of Illinois–Chicago) for her technical support with the electron microscopy.
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